Application of CD47 functional mimetic peptide in the preparation of M2 macrophage targeted delivery system

Abstract

The present invention relates to the application of CD47 functional mimetic peptide in the preparation of M2 macrophage targeted delivery system. The present invention discloses a strategy for combining "active targeting" and "active escape" to achieve highly selective M2 macrophage targeted delivery. M2 macrophages are important tumor treatment targets, and CD47 protein is a self-recognition protein in the body. After interacting with SIRPα protein on macrophages, it can inhibit the phagocytosis of macrophages, but the response of CD47 protein in M1 macrophages and M2 macrophages is not the same, and it can more significantly inhibit the phagocytosis of M1 macrophages, thereby improving the selectivity for M2 macrophages through the active escape of M1 macrophages. The present invention modifies CD47 functional mimetic peptide and M2 macrophage targeting ligand on the surface of nanocarrier liposomes, and the liposomes can be significantly taken up by M2 macrophages, but have no significant effect on M1 macrophages.

Description

Technical field:

[0001] The invention relates to application of CD47 function mimetic peptide in preparing M2 macrophage targeted delivery system, belonging to the technical field of biological targeted drugs. Background technology:

[0002] Tumor-associated macrophages (TAM) refer to macrophages that infiltrate surrounding tumor tissues and are derived from tissue-resident macrophages and bone marrow-derived monocytes. Tumor microenvironment (TME) refers to the surrounding microenvironment where tumor cells exist, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, various signaling molecules, and extracellular matrix (ECM) [Fukuda K, Kobayashi A, Watabe K. The role of tumor-associated macrophage in tumor progression [J]. Front Biosci (Schol Ed), 2012, 4: 787-798.]. Under the influence of TME, TAMs will polarize into classically activated M1 macrophages and alternatively activated M2 macrophages. Among these two types of macrophages, M1 macrophages have pro-inflammatory effects and can secrete pro-inflammatory factors to inhibit tumor growth, while M2 macrophages can promote tumor growth and metastasis and invasion by secreting transforming growth factor and vascular endothelial growth factor. Therefore, M1 macrophages can inhibit tumor growth, while M2 macrophages promote tumor growth [Wu K, Lin K, Li X, et al. Redefining Tumor-Associated macrophage subpopulations and functions in the tumor microenvironment [J]. Front Immunol, 2020, 11: 1731.]. In clinical diagnosis, the ratio of M1 macrophages to M2 macrophages in TME is closely related to the prognosis of tumor treatment. Generally speaking, the proportion of M1 macrophages is higher in the early stage of tumor, and the proportion of M2 macrophages is higher in the late stage of tumor.In summary, M2 macrophages are important targets for tumor treatment [Ding P, Wang W, Wang J, et al. Expression of tumor-associated macrophage in progression of human glioma[J]. Cell Biochem Biophys, 2014, 70(3): 1625-1631; Mantovani A, Marchesi F, Malesci A, et al. Tumour-associated macrophages as treatment targets in oncology[J]. Nat Rev Clin Oncol, 2017, 14(7): 399-416.].

[0003] The microenvironment in TME is a necessary condition for maintaining the tumor growth promoted by M2 macrophages. At present, some schemes have been proposed to target M2 macrophages for tumor treatment, including: 1) inhibiting macrophage recruitment, 2) reprogramming M2 macrophages into M1 macrophages, and 3) killing M2 macrophages. It is worth noting that in the latter two schemes, it is necessary to specifically target M2 macrophages. In order to improve the selectivity for M2 macrophages, the strategy of active targeted delivery is generally adopted, that is, according to the receptors highly expressed on M2 macrophages, the corresponding ligands are modified on the surface of the carrier, and the delivery efficiency is improved through active targeted recognition between ligands and receptors. Ideally, active targeted delivery should only interact with M2 macrophages. However, in actual situations, receptors highly expressed on M2 macrophages are generally also significantly expressed on M1 macrophages. Therefore, drugs targeting M2 macrophages will also be targeted and delivered to M1 macrophages. How to achieve precise selection of targeted drugs in M1 macrophages and M2 macrophages remains an important challenge in drug delivery [Binnemars-Postma K, Storm G, Prakash J. Nanomedicine strategies to target Tumor-Associated macrophages [J]. Int J Mol Sci, 2017, 18 (5): 979.].

[0004] CD47 is a glycoprotein expressed on all mammalian cells. After interacting with SIRPα on macrophages, it can trigger the "don't-eat-me" signaling pathway and inhibit the phagocytosis of macrophages [Oldenborg PA, Zheleznyak A, Fang YF, et al. Role of CD47 as a marker of self on red blood cells[J]. Science, 2000, 288(5473): 2051-2054; Rodriguez PL, Harada T, Christian DA, et al. Minimal "Self" Peptides That Inhibit Phagocytic Clearance and Enhance Delivery of Nanoparticles[J]. Science, 2013, 339(6122): 971-975.]. In the applicant's previous research, the applicant designed and constructed a new "Self" peptide that mimics the function of CD47, and explored its application in the long circulation of drugs in vivo [Tang Y, Wang X, Li J, et al. Overcoming the reticuloendothelial system barrier to drug delivery with a "Don't-Eat-Us" strategy [J]. ACS Nano, 2019, 13 (11): 13015-13026.]. However, there is still no relevant research and report on the precise selection of targeted drugs in M1 macrophages and M2 macrophages from the interaction between CD47 and SIRPα. Summary of the invention:

[0005] In view of the deficiencies of the prior art, the present invention provides the use of CD47 functional mimetic peptides in the preparation of M2 macrophage targeted delivery systems. In the previous study, the present invention confirmed that the response of the "don't-eat-me" signaling pathway combined with CD47 functional mimetic peptides and SIRPα is different in M1 macrophages and M2 macrophages. Compared with M2 macrophages, the phagocytic ability of M1 macrophages is more significantly inhibited by the "don't-eat-me" signaling pathway. In combination with this finding, the present invention improves the recognition ability of targeted drugs in M1 / M2 macrophages to M2 macrophages by combining active escape ("don't-eat-me" signal) with active targeting ("eat-me" signal).

[0006] The technical solution of the present invention is as follows:

[0007] Use of a CD47 function mimicking peptide in the preparation of an M2 macrophage targeted delivery system, wherein the amino acid sequence of the CD47 function mimicking peptide is shown in SEQ ID NO.1, i.e., GNYTCEVTELSREGKTVIELK.

[0008] The CD47 functional mimetic peptide (Self peptide) of the present invention is a functional sequence of CD47 screened out according to a molecular docking simulation experiment.

[0009] Preferably according to the present invention, the M2 macrophage targeted delivery system comprises a CD47 function mimicking peptide and a targeting ligand for M2 macrophages.

[0010] Further preferably, the targeting ligand for M2 macrophages is galactose.

[0011] Further preferably, the CD47 function-mimicking peptide mediates the "active escape" of M1 macrophages, inhibiting the phagocytosis of M1 macrophages; the targeting ligand mediates the "active targeting" of M2 macrophages, promoting the targeting and phagocytosis of M2 macrophages.

[0012] Preferably according to the present invention, the M2 macrophage targeted delivery system is a liposome targeted delivery system.

[0013] Further preferably, the main components of the liposome material of the liposome targeted delivery system are egg yolk phosphatidylcholine and cholesterol.

[0014] Further preferably, the molar ratio of egg yolk phosphatidylcholine to cholesterol in the liposome material is 1-5:1, more preferably 2-3:1.

[0015] In the present invention, the main components of the liposome material are egg yolk lecithin and cholesterol. Egg yolk lecithin is a molecule composed of glycerol, fatty acids, phosphoric acid, etc. The phospholipid "tail" is hydrophobic, and the fatty acid "head" is hydrophilic. The fatty acids are respectively modified at the amino group of galactose and the amino terminal of the CD47 functional mimetic peptide. Therefore, when preparing liposomes, fatty acids can be inserted into the hydrophobic end of phospholipids, so that the ligands (galactose and "Self" peptide) can be modified on the surface of the liposomes.

[0016] Further preferably, the molar percentage of the targeting ligand to the liposome material is less than 1%, and the molar percentage of the CD47 function mimetic peptide to the liposome material is more than 1%.

[0017] More preferably, the molar percentage of the targeting ligand to the liposome material is 0.5% to 1%, and the molar percentage of the CD47 function mimetic peptide to the liposome material is 1% to 3%.

[0018] An M2 macrophage targeted delivery system comprises a CD47 function mimicking peptide and a M2 macrophage targeting ligand, wherein the amino acid sequence of the CD47 function mimicking peptide is as shown in DEQ ID NO.1, i.e., GNYTCEVTELSREGKTVIELK.

[0019] Preferably according to the present invention, the targeting ligand for M2 macrophages is galactose.

[0020] Preferably according to the present invention, the M2 macrophage targeted delivery system is a liposome targeted delivery system.

[0021] Further preferably, the main components of the liposome material of the liposome targeted delivery system are egg yolk phosphatidylcholine and cholesterol.

[0022] Further preferably, the molar ratio of egg yolk phosphatidylcholine to cholesterol in the liposome material is 1-5:1, more preferably 2-3:1.

[0023] Further preferably, the molar percentage of the targeting ligand to the liposome material is less than 1%, and the molar percentage of the CD47 function mimetic peptide to the liposome material is more than 1%.

[0024] More preferably, the molar percentage of the targeting ligand to the liposome material is 0.5% to 1%, and the molar percentage of the CD47 function mimetic peptide to the liposome material is 1% to 3%.

[0025] Preferably, according to the present invention, the M2 macrophage targeted delivery system is prepared according to the following method: using methanol as a solvent to prepare a targeting ligand solution, a CD47 functional mimetic peptide solution and an egg yolk lecithin solution, respectively, and using chloroform as a solvent to prepare a cholesterol solution; mixing the egg yolk lecithin solution and the cholesterol solution in proportion, and then adding the CD47 functional mimetic peptide solution and the targeting ligand solution in proportion, decompressing and drying the above mixed solution, hydrating it with PBS buffer, shaking it in a 37°C water bath, sonicating it with a probe sonicator and extruding it through a liposome extruder with a 100nm polycarbonate membrane, to obtain liposomes modified with the targeting ligand and the CD47 functional mimetic peptide, which is the M2 macrophage targeted delivery system.

[0026] A drug-encapsulated M2 macrophage targeted delivery system is a drug-encapsulated M2 macrophage targeted delivery system in which a disease treatment drug is carried.

[0027] Preferably according to the present invention, the drug is doxorubicin.

[0028] In a preferred technical solution of the present invention, the M2 macrophage targeted delivery system encapsulated doxorubicin (DOX), the half inhibitory concentration (IC50) of DOX in M1 macrophages is 0.91 μg / mL, and the half inhibitory concentration (IC50) in M2 macrophages is 0.19 μg / mL. The half inhibitory concentration of M1 macrophages is increased by about 4.8 times compared with that of M2 macrophages, and the effect difference is significant.

[0029] The technical features of the present invention are as follows:

[0030] The present invention relates to a strategy for combining "active targeting" and "active escape" to achieve highly selective M2 macrophage targeted delivery. In tumors and other sites, tumor-associated macrophages can differentiate into classically activated M1 macrophages and alternatively activated M2 macrophages. Among them, M1 macrophages can release proinflammatory factors to exert tumor-suppressing effects, while M2 macrophages can secrete transforming growth factor and vascular endothelial growth factor to promote tumor growth, proliferation and migration. Therefore, M2 macrophages are important tumor treatment targets. At present, in order to improve the lethality against M2 macrophages, the corresponding ligands are often modified on the surface of nanocarriers according to the upregulated receptors on M2 macrophages, and active targeting (eat-me signal) is achieved through the recognition between ligands and receptors to improve the lethality of nano drugs to M2 macrophages. However, in practical applications, the upregulated receptors of M2 macrophages are also significantly expressed on common tissue-resident macrophages such as M1 macrophages and liver and spleen macrophages. Therefore, this targeted delivery strategy lacks sufficient selectivity between different macrophage phenotypes. CD47 protein is a self-recognition protein in the body. After interacting with SIRPα protein on macrophages, it can trigger the "don't-eat-me" signal and inhibit the phagocytosis of macrophages (active escape), but the response of CD47 protein in M1 macrophages and M2 macrophages is not the same. Between the two, the "don't-eat-me" signal response can more significantly inhibit the phagocytosis of M1 macrophages, thus providing a way to improve the selectivity for M2 macrophages through the active escape of M1 macrophages. In this invention, the applicant modified the CD47 functional mimetic peptide-"Self" peptide and the M2 macrophage targeting ligand-galactose on the surface of the nanocarrier liposomes, respectively, and confirmed that after the ratio was optimized, the liposomes could be significantly taken up by M2 macrophages, but had no significant effect on M1 macrophages. The half maximal inhibitory concentration (IC50) of the liposomes encapsulating doxorubicin as a model drug on M1 macrophages was about 4.8 times higher than that on M2 macrophages, confirming that this strategy significantly improved the selectivity for M2 macrophages.

[0031] Beneficial effects of the present invention:

[0032] The present invention has found through research that the response of the "don't-eat-me" signaling pathway of the CD47 functional mimic peptide-"Self" peptide and SIRPα combined is different in M1 macrophages and M2 macrophages. Compared with M2 macrophages, the phagocytic ability of M1 macrophages is more significantly inhibited by the "don't-eat-me" signaling pathway. Based on the high expression of galactose C-terminal lectin on the surface of M2 macrophages, the present invention synthesized N-oleoylgalactose as an active targeting ligand, and modified N-oleoylgalactose (G) and palmitic acid-modified "Self" peptide (S) on the surface of liposomes. The present invention shows that liposomes (S-Lip) modified with "Self" peptides at a molar percentage of more than 1% can significantly inhibit the endocytosis rate of M1 macrophages; qualitative and quantitative analysis of cell uptake shows that the uptake enhancement of liposomes (GS-Lip) modified with "Self" peptides and galactose in M2 macrophages is significantly higher than that in M1 macrophages, and when the galactose modification amount is below 1%, GS-Lip significantly improves the uptake of liposomes by M2 macrophages while not significantly affecting the "active escape" of M1 macrophages. Using doxorubicin hydrochloride as a liposome model drug, cytotoxicity experiments show that the half-inhibitory concentration of GS-Lip loaded with doxorubicin hydrochloride for M1 macrophages is about 4.8 times higher than that of M2 macrophages. Therefore, the targeted drug delivery system of the present invention successfully distinguishes between M1 macrophages and N2 macrophages, and significantly improves the targeting selectivity for M2 macrophages. Description of the drawings:

[0033] Figure 1 The flow cytometric results of M1 macrophage induction are shown in Figure 2.

[0034] Figure 2 The flow cytometric results show the induction of M2 macrophages.

[0035] Figure 3 This is the electrophoresis analysis of tyrosine phosphorylation of SIRPα induced by co-incubation of "Self" peptide-liposomes (S-Lip) with M1 macrophages and M2 macrophages. The activation of this phosphorylation pathway can inhibit the phagocytic function of macrophages.

[0036] Figure 4 It is a line graph of the endocytosis ratio of M1 macrophages to “Self” peptide-liposomes (S-Lip) with different amounts of “Self” peptide modification.

[0037] Figure 5 Fluorescence imaging photos of M2 macrophages taking up galactose-"Self" peptide-liposomes (GS-Lip) with different galactose modification amounts.

[0038] Figure 6 The effect of galactose-"Self" peptide-liposome (GS-Lip) loaded with doxorubicin on the survival rate of M1 / M2 macrophages. Specific implementation method:

[0039] The technical solution of the present invention will be described in detail below in conjunction with the embodiments and drawings, but the protection scope of the present invention is not limited thereto.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0041] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0042] Example 1: Induction of M1 / M2 macrophages

[0043] RAW264.7 cells of the M0 mouse macrophage cell line in the logarithmic growth phase (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were cultured at a cell number of 5×10 5 The cells were inoculated into 12-well plates and cultured for 24 h.

[0044] Induction of M1 macrophages: Lipopolysaccharide (LPS, Sigma) and IFNγ (Biyuntian Biotechnology) were added to the RAW264.7 cell culture well plate, with the final concentration of LPS being 100 ng / mL and the final concentration of IFNγ being 25 ng / mL, and the culture was continued for 24 h; digestion, counting, and then staining with fluorescently labeled anti-CD86 (M1 macrophage marker protein), fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and then stained with fluorescently labeled anti-CD206 (M2 macrophage marker protein), and the cell population was analyzed by flow cytometry (BD FACS Calibur). The results of flow cytometry analysis are shown in Figure 2. Figure 1 As shown, about 77.7% of RAW264.7 cells were induced to become M1 macrophages.

[0045] Induction of M2 macrophages: IL-4 was added to the RAW264.7 cell culture plate to a final concentration of 10 ng / mL, and the cells were cultured for 24 h. The cells were digested, counted, and then stained with fluorescently labeled anti-CD86 (a marker protein for M1 macrophages), fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and then stained with fluorescently labeled anti-CD206 (a marker protein for M2 macrophages). The cell population was analyzed by flow cytometry (BD FACS Calibur). The results of flow cytometry analysis are shown in Figure 2. Figure 2As shown, about 49.8% of RAW264.7 cells were induced to become M2 macrophages.

[0046] Example 2: Differences in the response of the “don't-eat-me” signaling pathway of CD47 functional mimetic peptide-“Self” peptide in M1 macrophages and M2 macrophages

[0047] The amino acid sequence of the CD47 functional mimetic peptide - "Self" peptide is shown in SEQ ID NO.1, i.e., GNYTCEVTELSREGKTVIELK, which is obtained by artificial synthesis, and palmitic acid is added to the amino segment by solid phase synthesis during the synthesis [Cao L, Fischer A, Bornscheuer UT, et al. Lipase-Catalyzed Solid Phase Synthesis of Sugar Fatty Acid Esters. Biocatalysis, 1996, 14(4): 15.].

[0048] The preparation method of the “Self” peptide-modified liposomes is as follows:

[0049] Use methanol as solvent to prepare 1 mg / mL of "Self" peptide solution and 40 mg / mL of egg yolk lecithin (PL-100m, purchased from Avit) solution, and use chloroform as solvent to prepare 10 mg / mL of cholesterol (injection grade, purchased from Avit) solution; according to the molar ratio of egg yolk lecithin to cholesterol of 7:3, the egg yolk lecithin solution and the cholesterol solution are mixed with a volume ratio of about 4.5:1, and then the "Self" peptide solution is added, and the molar percentage of "Self" peptide to liposome membrane material (egg yolk lecithin and cholesterol) is 1%. The above mixed solution was dried under reduced pressure, and trace organic solvents were removed by nitrogen; evaporated to dryness under reduced pressure to obtain an egg yolk lecithin film, and nitrogen was passed to remove trace organic solvents; PBS solution (pH = 7.2) was added to the egg yolk lecithin film, shaken in a 37°C water bath, sonicated with a probe sonicator and extruded through a liposome extruder with a 100nm polycarbonate membrane to obtain "Self" peptide-modified liposomes (S-Lip), which were stored for later use.

[0050] The polarized M1 macrophages and M2 macrophages induced in Example 1 were inoculated in 6 cm culture dishes, respectively, and incubated in DMEM culture medium (containing 10% fetal bovine serum) at 37°C; S-Lip and DMEM culture medium (containing 10% fetal bovine serum) were taken and pre-incubated at 37°C for 30 minutes, then the original culture medium in the culture dish was aspirated and discarded, and the culture dish was washed 3 times with pre-warmed (37°C) PBS buffer (pH=7.2), and then the DMEM culture medium solution containing S-Lip was added, and the final concentration of S-Lip was 500 μg / mL, and the culture dish was incubated at 37°C for 1 hour. The culture dish was transferred to an ice bath, 500 μL RIPA cell lysis buffer (containing 1% PMSF and 1% protein phosphatase inhibitor) (purchased from Beijing Dingguo Biology) was added, and the cell lysis solution was left to stand for 1 hour, and then the cell lysate was transferred to a 2 mL centrifuge tube, centrifuged at 10000 rpm and 4°C for 10 minutes, and the supernatant was transferred to a new centrifuge tube, and the precipitate was discarded. Add 0.5 μg SIRPα antibody (purchased from Wuhan Sanying Biotechnology Co., Ltd.) to the supernatant and shake slowly overnight (16 h) at 4°C. Resuspend Protein A+G Agarose (purchased from Biyuntian Biotechnology) by pipetting, add 20 μL to the supernatant, and shake in an ice bath for 4 h. Centrifuge at 4°C, 2500 rpm for 10 min to fully precipitate Protein A+G Agarose, and remove the supernatant. Resuspend the Protein A+G Agarose precipitate with 1 mL RIPA cell lysis buffer (containing 1% PMSF and 1% protein phosphatase inhibitors), then continue centrifugation, discard the supernatant, and repeat the operation 5 times to wash completely. Resuspend the precipitate with 50 μL 2× electrophoresis loading buffer (Beijing Dingguo Biotechnology), boil in water for 10 min, centrifuge at 4°C, 10000 rpm for 10 min, and take the supernatant for loading. The same amount of sample was loaded twice, and membrane A and membrane B were obtained after electrophoresis and transfer. Among them, membrane A was incubated with SIRPα antibody (1:500 dilution) (purchased from Wuhan Sanying Biotechnology Co., Ltd.), and membrane B was incubated with tyrosine phosphorylation antibody (1:100 dilution) (purchased from Boster Biotechnology Co., Ltd.). After incubation with secondary antibody (HRP labeled) (purchased from Bioson Biotechnology Co., Ltd.), the membrane was developed and analyzed by GE Amersham Imager 600 (see Figure 3 ).

[0051] The results are as follows Figure 3 As shown, after S-Lip was incubated with macrophages respectively, the tyrosine phosphorylation level of SIRPα in M1 macrophages was significantly higher than that in M2 macrophages. The activation of this phosphorylation pathway can inhibit the phagocytic function of macrophages, confirming that the "don't-eat-me" signal induced by the interaction between the "Self" peptide and SIRPα is more inclined to inhibit the phagocytic function of M1 macrophages, thereby achieving the "active escape" of M1 macrophages.

[0052] Example 3: Optimization of the modification amount of CD47 functional mimetic peptide-"Self" peptide

[0053] The preparation method of the “Self” peptide-modified liposomes is as follows:

[0054] 1 mg / mL of "Self" peptide solution and 40 mg / mL of egg yolk lecithin (PL-100m, purchased from Avit) solution were prepared using methanol as solvent, and 10 mg / mL of cholesterol (injection grade, purchased from Avit) solution was prepared using chloroform as solvent; the egg yolk lecithin solution and the cholesterol solution were mixed according to a molar ratio of 7:3 between egg yolk lecithin and cholesterol, with a volume ratio of about 4.5:1, and then the "Self" peptide solution was added, and the molar percentages of the "Self" peptide and the liposome membrane material (egg yolk lecithin and cholesterol) were set to 0.1%, 0.5%, 1%, and 3%, respectively. The above mixed solutions were respectively dried under reduced pressure and nitrogen was used to remove trace organic solvents; evaporated to dryness under reduced pressure to obtain an egg yolk lecithin film, and nitrogen was passed to remove trace organic solvents; 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) was dissolved in PBS buffer (pH=7.2), and then added to the egg yolk lecithin film, the final HPTS concentration was 10mM, oscillated in a 37°C water bath, sonicated with a probe sonicator and extruded through a liposome extruder with a 100nm polycarbonate membrane, and then dialyzed in a PBS buffer (pH=7.2) with a dialysis bag (molecular weight cutoff 8000-14000Da) to remove free HPTS, and HPTS-labeled "Self" peptide-modified liposomes (S-Lip) were obtained, which were stored for later use.

[0055] 8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) is a water-soluble pH probe that cannot pass through the cell membrane by itself and has different fluorescence spectra under acidic conditions in the cytoplasm and neutral conditions in the cell culture medium.

[0056] The prepared HPTS-labeled S-Lip and liposomes (Lip) without "Self" peptide modification were added to the M1 macrophage culture wells, and samples were taken at different times according to relevant literature. The multifunctional microplate reader was used for analysis. The setting mode was fluorescence intensity detection, emission wavelength: 510nm, excitation wavelength: 400-480nm, and the calculation method was:

[0057]

[0058] Among them, the ratio represents the ratio of the fluorescence intensity at the excitation wavelength of 450nm and 413nm (450nm / 413nm), the ratio (pH7.4) represents the ratio of the fluorescence intensity of S-Lip or Lip at the excitation wavelength of 450nm and 413nm in PBS buffer at pH7.4, and the ratio (pH6.0) represents the ratio of the fluorescence intensity of S-Lip or Lip at the excitation wavelength of 450nm and 413nm in acidic buffer at pH6.0.

[0059] The analysis results are as follows Figure 4 The liposomes (Lip) without "Self" peptide modification had the highest endocytosis rate in M1 macrophages. The liposomes (S-Lip) modified with "Self" peptide, "Self" peptide bound to SIRPα on the surface of M1 macrophages, sending out a "don't-eat-me" signal, weakening the phagocytic effect of M1 macrophages and decreasing the endocytosis rate. As the amount of "Self" peptide modification increased, the endocytosis rate decreased. When the amount of "Self" peptide modification was above 1%, the endocytosis of S-Lip by M1 macrophages was significantly inhibited.

[0060] Example 4: Optimization of the modification amount of galactose

[0061] Galactose and "Self" peptide modified liposomes were prepared as follows:

[0062] The synthesis of N-oleoylgalactose was carried out according to the literature [Yan LA, By B, Xz A. Oral delivery of imatinib through galactosylated polymeric nanoparticles to explore the contribution of a saccharide ligand to absorption-Science Direct [J]. International Journal of Pharmaceutics, 568: 118508-118508.].

[0063] 1 mg / mL N-oleoylgalactose solution, 1 mg / mL "Self" peptide solution and 40 mg / mL egg yolk lecithin (PL-100m, purchased from Avit) solution were prepared with methanol as solvent, and 10 mg / mL cholesterol (injection grade, purchased from Avit) solution was prepared with chloroform as solvent; the egg yolk lecithin solution and the cholesterol solution were mixed according to the molar ratio of egg yolk lecithin to cholesterol of 7:3, with a volume ratio of about 4.5:1, and then the "Self" peptide solution was added, and the molar percentage of the "Self" peptide to the liposome membrane material (egg yolk lecithin and cholesterol) was 1%; then the N-oleoylgalactose solution was added, and the molar percentages of N-oleoylgalactose to the liposome membrane material (egg yolk lecithin and cholesterol) were set to 0.5%, 1%, and 2%, respectively; then about 40 μg Lissrhod pe EPC phospholipids (Avanti) were fluorescently labeled; the mixed solution was dried under reduced pressure, hydrated with PBS buffer (pH = 7.2), shaken in a 37°C water bath, sonicated with a probe sonicator and extruded through a liposome extruder with a 100 nm polycarbonate membrane to obtain fluorescently labeled galactose and "Self" peptide-modified liposomes (GS-Lip), which were stored for later use.

[0064] Activated M1 macrophages and M2 macrophages were seeded into 96-well plates at 2000 cells / well, respectively. After overnight culture, different fluorescently labeled GS-Lip, fluorescently labeled unmodified liposomes (Lip), and fluorescently labeled "Self" peptide-modified liposomes (1% S-Lip) were added, respectively. After incubation for 2 hours, the plates were washed with PBS buffer (pH=7.2), fixed with 4% paraformaldehyde, and then stained with DAPI (5μg / mL) and observed with a high-content cell imaging system.

[0065] The results are as follows Figure 5 , where blue represents the cell nucleus and red represents the liposome. There is no significant difference in the endocytosis of liposomes (Lip) without any modification in M1 macrophages and M2 macrophages, and both show a certain degree of endocytosis. The liposomes modified with "Self" peptide (1% S-Lip) did not show red fluorescence in M1 macrophages, and the endocytosis phenomenon disappeared, but there was no obvious change in M2 macrophages, and the red fluorescence still existed, which also verified that the response of the "don't-eat-me" signaling pathway combined with "Self" peptide and SIRPα is different in M1 macrophages and M2 macrophages. Liposomes modified with galactose and "Self" peptide (GS-Lip) can significantly enhance the uptake of liposomes by M2 macrophages, but only when the amount of galactose modification is below 1%, GS-Lip will not significantly affect M1 macrophages while significantly improving the uptake of liposomes by M2 macrophages.

[0066] Embodiment 5:

[0067] Galactose and "Self" peptide modified liposomes were prepared as follows:

[0068] 1 mg / mL N-oleoylgalactose solution, 1 mg / mL "Self" peptide solution and 40 mg / mL egg yolk lecithin (PL-100m, purchased from Avit) solution were prepared with methanol as solvent, and 10 mg / mL cholesterol (injection grade, purchased from Avit) solution was prepared with chloroform as solvent; the egg yolk lecithin solution and the cholesterol solution were mixed according to the molar ratio of egg yolk lecithin to cholesterol of 7:3, with a volume ratio of about 4.5:1; then the "Self" peptide solution and N-oleoylgalactose solution were added, and the molar percentage of "Self" peptide and N-oleoylgalactose to liposome membrane material (egg yolk lecithin and cholesterol) was 1%; the above mixed solution was decompressed and dried by spin drying, and hydrated with 250 mM ammonium persulfate solution, The mixture was shaken in a 37°C water bath, sonicated with a probe sonicator and extruded through a liposome extruder with a 100 nm polycarbonate membrane to obtain blank galactose and "Self" peptide-modified liposomes; the blank galactose and "Self" peptide-modified liposomes were dialyzed with pure water for 6 h (the molecular weight cutoff of the dialysis bag was 8000-14000 Da) to remove free ammonium persulfate, and then doxorubicin hydrochloride was dissolved in pure water, the doxorubicin hydrochloride solution was added to the liposome solution, incubated in a 37°C water bath for 1 h, and then continued to dialyze with a dialysis bag (the molecular weight cutoff of the dialysis bag was 8000-14000 Da) to prepare galactose and "Self" peptide-modified liposomes (DOX-GS-Lip) encapsulating doxorubicin hydrochloride, and placed at 4°C for use.

[0069] The induced activated M1 macrophages and M2 macrophages were seeded in 96-well plates at 5000 cells / well, respectively, and cultured overnight until they were completely attached to the wall; liposomes (DOX-GS-Lip) modified with galactose and "Self" peptide with different doxorubicin hydrochloride contents were added, and the doxorubicin hydrochloride content in the liposomes was re-determined after the preparation of DOX-GS-Lip, and the incubation was continued for 24 hours; the culture medium was removed, MTT was added to a final concentration of 0.5 mg / mL, incubated for 4 hours, the supernatant was removed, 150 μL DMSO was added to each well, and incubated at 37°C for 10 minutes until the purple crystals were completely dissolved, the absorbance was detected by an enzyme reader at 450 nm, and the relative cell survival rate was analyzed.

[0070] The results are as follows Figure 6As shown, under the conditions of different doxorubicin hydrochloride contents, the cell survival rate of M1 macrophages was higher than that of M2 macrophages, and the half inhibitory concentration (IC50) of DOX in M1 macrophages and M2 macrophages was 0.91 μg / mL and 0.19 μg / mL, respectively, and there was an obvious selective difference between the two. SEQUENCE LISTING <110> Shandong First Medical University (Shandong Academy of Medical Sciences) Institute of Materia Medica, Shandong Academy of Medical Sciences (Shandong Anti-Aging Research Center, Shandong New Technology Pharmaceutical Research Institute) <120> Application of CD47 functional mimetic peptide in the preparation of M2 macrophage targeted delivery system <160> 1 <170> PatentIn version 3.5 <210> 1 <211> twenty one <212> PRT <213> Artificial sequence <400> 1 Gly Asn Tyr Thr Cys Glu Val Thr Glu Leu Ser Arg Glu Gly Lys Thr 1 5 10 15 Val Ile Glu Leu Lys 20

Claims

1. Application of a CD47 functional mimetic peptide in the preparation of an M2 macrophage targeted delivery system, the M2 macrophage targeted delivery system comprising a CD47 functional mimetic peptide and a targeting ligand for M2 macrophages, the amino acid sequence of the CD47 functional mimetic peptide being as shown in SEQ ID NO.1, i.e., GNYTCEVTELSREGKTVIELK, the M2 macrophage targeted delivery system being capable of promoting the targeting and phagocytosis of M2 macrophages, and inhibiting the phagocytosis of M1 macrophages, the targeting ligand for M2 macrophages being galactose, the M2 macrophage targeted delivery system being a liposome targeted delivery system, the molar percentage of the targeting ligand to the liposome material being 0.5% to 1%, and the molar percentage of the CD47 functional mimetic peptide to the liposome material being 1% to 3%.

2. The use according to claim 1, characterized in that The liposome materials of the liposome targeted delivery system are egg yolk lecithin and cholesterol.

3. The use according to claim 2, characterized in that The molar ratio of egg yolk phosphatidylcholine to cholesterol in the liposome material is 1-5:

1.

4. The use according to claim 3, characterized in that The molar ratio of egg yolk phosphatidylcholine to cholesterol in the liposome material is 2-3:

1.

5. An M2 macrophage targeted delivery system, characterized in that: It comprises a CD47 function simulation peptide and a targeting ligand for M2 macrophages, wherein the amino acid sequence of the CD47 function simulation peptide is as shown in DEQ ID NO.1, i.e., GNYTCEVTELSREGKTVIELK, and the targeting ligand for M2 macrophages is galactose; the M2 macrophage targeting delivery system is a liposome targeting delivery system, wherein the liposome materials are egg yolk lecithin and cholesterol, and the molar ratio of egg yolk lecithin to cholesterol is 1-5:

1.

6. The M2 macrophage targeted delivery system according to claim 5, characterized in that: The molar ratio of the egg yolk lecithin to cholesterol is 2-3:

1.

7. The M2 macrophage targeted delivery system according to claim 5, characterized in that: The molar percentage of the targeting ligand to the liposome material is 0.5% to 1%, and the molar percentage of the CD47 functional mimetic peptide to the liposome material is 1% to 3%.

8. The M2 macrophage targeted delivery system according to claim 5, characterized in that: The M2 macrophage targeted delivery system is prepared according to the following method: using methanol as a solvent to prepare a targeting ligand solution, a CD47 functional mimetic peptide solution and an egg yolk lecithin solution respectively, and using chloroform as a solvent to prepare a cholesterol solution; mixing the egg yolk lecithin solution and the cholesterol solution according to a proportion, and then adding the CD47 functional mimetic peptide solution and the targeting ligand solution according to a proportion; decompressing and spinning the above mixed solution, hydrating it with PBS buffer, shaking it in a 37°C water bath, sonicating it with a probe sonicator, and extruding it through a liposome extruder with a 100nm polycarbonate membrane to obtain liposomes modified with the targeting ligand and the CD47 functional mimetic peptide, that is, the M2 macrophage targeted delivery system.

9. A drug-loaded M2 macrophage targeted delivery system, characterized in that: The disease treatment drug is carried in the M2 macrophage targeted delivery system described in claim 5.

10. The drug-loaded M2 macrophage targeted delivery system according to claim 9, characterized in that: The drug is doxorubicin.

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